(278e) Multi-Zonal Three-Dimensional in Vitro Culture Model of Growth Plate Cartilage Using Alginate Hydrogel Scaffolds

Organ regeneration using engineered tissues has the potential to revolutionize personalized patient care. However, precisely controlling the rate and direction of tissue growth remains a significant challenge for tissue engineers. Advanced tissue culture models that enable investigation of the mechanisms underlying anisotropic growth are necessary to overcome this challenge. During skeletal development, cartilaginous growth plates at epiphyses drive polarized bone growth, or endochondral ossification. This process is controlled by the precise regulation of chondrocyte differentiation via long- and short-range signaling mechanisms within the growth plateâ??s architecture. As a result, the growth plate is organized in morphologically unique zones, each containing chondrocytes with unique phenotypes. While much has been described about the process of development of the growth plate, there is still a gap in the understanding of the interplay between specific signaling mechanisms and resulting architecture. To date, not enough is known about these intricacies to effectively modulate chondrocyte maturation in existing in vitro cartilage culture models. The design of an in vitro culture model of growth plate cartilage will be utilized to elucidate the structure and function of the regulatory network that coordinates chondrocyte maturation and leads to the formation of zonal architecture. This project focuses on the utilization of alginate hydrogels in the design of a novel, multi-zonal, three-dimensional (3-D) in vitro culture system aiming to recapitulate the supportive extracellular matrix and diffusional gradients of signaling factors found in growth plate cartilage. In order to test the hypothesis that the activation of the hedgehog (HH) signaling pathway is necessary for zonal arrangement, and in particular the formation of the columnar structures found in the proliferative zone of growth plate cartilage, radially layered alginate bead constructs are employed. We first show that an alginate bead culture method is supportive of cell viability and conducive to the presentation of factors known to modulate HH signaling, such as parathyroid hormone (PTH/PTHrP) and Indian Hedgehog (IHH), and the subsequent response of primary mouse growth plate chondrocytes to these factors. This response was observed with flow cytometry, ddPCR and western blot to monitor HH signaling. In order to present these factors in a gradient, a layered bead construct was designed and optimized for the controlled response of chondrocytes to signaling factors. HEK293T cells transfected with a plasmid to produce PTH were encapsulated in an innermost alginate bead, while growth plate chondrocytes were encapsulated in an alginate layer around this inner bead, receiving PTH in a radially diffusing fashion. IHH was added to the media to set up a bidirectional diffusional gradient. The response of the chondrocytes to this gradient and the resulting zonal arrangement of the growth plate chondrocytes was monitored by the above methods along with the use of Fluorescent In Situ Hybridization (FISH) to visualize the characteristic, radially differential expression of genes specific to each of the defined zones. This scaffold design has proven to be a powerful tool in investigating the role of these specific factors on zonal development in the growth plate. With the knowledge gained from these experiments, we begin to form a better picture of the mechanisms behind growth plate cartilage maturation, which will lead to the development of better therapeutic techniques for patients suffering from growth plate cartilage disease or injury.